The overall objective is to provide chemical methods for the sequence specific cleavage of genomic DNA which afford a broad range of new specificities with higher sequence specificity than is currently available with restriction enzymes. Pyrimidine oligonucleotides bind duplex DNA sequence specifically at purine sites to form a triple helix structure. The pyrimidine oligomer is oriented in the major groove of DNA parallel to the Watson-Crick purine strand by Hoogsteen base pairing. Oligonucleotides, 15-18 bases in length, and equipped with a DNA cleaving function EDTA-Fe at the 5' end, cause sequence specific double strand breaks at single sites in plasmid DNA 4 kbp and lambdal bacteriophage genome (48.5 kbp). Due to the length of the recognition site, in a formal sense, this is 106 times more sequence specific than restriction enzymes. It is important to determine whether the full potential of this cleavage specificity can be realized. Because modified oligonucleotides can be synthesized by automated methods, this methodology affords a new class of "rare cutters" which could become readily available to the biological community. Specific objectives are to (1) optimize cleavage efficiency by the synthesis and study of oligonucleotide probes with multiple EDTA-Fe moieties attached, (2) analyze sequence specificity of oligonucleotide binding by studying the influence of pH, temperature, and organic cosolvent on probes of different base composition, (3) generalize a set of conditions for unique recognition at purine-DNA sites 15-18 base pairs in length, (4) define conditions for mapping DNA sites of partial homology, (5) analyze the effects of DNA target size on the kinetics of binding and measure binding affinities at discrete sites, (6) develop methods for raising the ratio of specific to non-specific binding by studying cooperatively binding oligonucleotides based on base stacking, and peptide-DNA probes, (7) design and synthesize novel oligonucleotides for mixed purine-pyrimidine sequences, (8) construct novel "crossover" oligonucleotides for binding palindromic homopurine sequences, (9) develop post-cleavage labeling methods at specific EDTA cleavage sites of genomic DNA, (10) investigate whether post-cleavage labeling could be made DNA fragment specific by recessed end hybridization.